Journal. .of. .General. .Virology. .(1998),. .79,. . 2411–2417.. . . . . Printed. .in. . .Great. . Britain. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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Pathogenesis of pneumovirus infections in mice : detection of
pneumonia virus of mice and human respiratory syncytial virus
mRNA in lungs of infected mice by in situ hybridization
Pamela M. Cook,1 Roger P. Eglin2 and Andrew J. Easton1
1
Department of Biological Sciences, University of Warwick, Coventry CV4 7AL, UK
2
Public Health Laboratory, Bridle Path, York Road, Leeds LS15 7TR, UK
The pathogenesis of pneumonia virus of mice (PVM) signs of infection. In animals which survived the
and human respiratory syncytial virus (HRSV) in infection virus-speciﬁc mRNA could not be detected
BALB/c mice were investigated by using in situ 10 days post-infection. Mice infected with
hybridization to detect virus mRNA in ﬁxed lung 1500 p.f.u. HRSV showed signiﬁcant differences in
sections. Following intranasal inoculation with the distribution of virus-speciﬁc mRNA when com-
120 p.f.u. PVM the pattern of hybridization showed pared to the pattern seen with PVM. HRSV mRNA
that virus mRNA was initially detected within 2 days was detected over large areas, but predominantly in
in alveolar cells. As the infection progressed the peribronchiolar and perivascular regions of the
number of hybridizing alveolar cells increased and lungs 5 days post-infection. The yield of PVM from
signal was also detected in cells lining the terminal infected mouse lungs was considerably higher than
bronchioles. By days 4 to 5 post-infection areas of that of HRSV. The possible implications of these
morphological abnormality could be seen, particu- results for the use of the mouse model for pneumo-
larly in the strongly hybridizing regions of the lung, virus infections are discussed.
and this correlated with the appearance of clinical
Introduction glutination-inhibiting response, though the clinical conse-
Pneumonia virus of mice (PVM) is a common pathogen in quences of human infection are unclear (Pringle & Eglin, 1986).
laboratory animal colonies, particularly those containing PVM was the ﬁrst described example of the viruses now
athymic mice (Homberger & Thomann, 1994 ; Kraft & Meyer, classiﬁed in the subfamily Pneumovirinae of the family Para-
1990). The virus was ﬁrst isolated from apparently healthy myxoviridae, which include human, bovine and ovine res-
animals and while the naturally acquired infection is thought to piratory syncytial viruses (HRSV, BRSV, ORSV) and avian
be asymptomatic, passage of virus in mice lungs resulted in pneumovirus (Pringle, 1996). Molecular cloning has shown
overt signs of disease ranging from an upper respiratory tract that the organization of the PVM genome is similar to that of
infection (Smith et al., 1984) to a fatal pneumonia (Carthew & HRSV (Chambers et al., 1990). Restricted serological cross-
Sparrow, 1980 a ; Richter et al., 1988 ; Weir et al., 1988). Despite reactivity between the nucleocapsid protein (N) and the
its name the natural host of PVM is not yet established and phosphoprotein (P) of PVM and HRSV has been described but
serological evidence indicates that many rodent species can be none of the external proteins show cross-reactivity, and the
infected (Eaton & van Herick, 1944 ; Horsfall & Curnen, 1946). viruses can be distinguished from each other in neutralization
In addition, the presence of neutralizing antibodies indicates assays (Gimenez et al., 1984 ; Ling & Pringle, 1989). While the
that some primates, including man, are infected with an amino acid sequence identity of some of the PVM and HRSV
antigenically indistinguishable virus (Horsfall & Curnen, 1946 ; proteins can be high, the nucleotide sequence identity between
Pringle & Eglin, 1986). Up to 80 % of serum samples taken from genes of PVM and RSVs is much lower and ranges from 60 %
human adults elicit a PVM-speciﬁc neutralizing and haemag- for the most highly conserved N gene (Barr et al., 1991) to the
nonstructural protein genes which show little or no identity
(Chambers et al., 1991). However, despite the high degree of
Author for correspondence : Andrew Easton. sequence conservation there is no cross-hybridization between
Fax j44 1203 523791. e-mail ae!dna.bio.warwick.ac.uk N genes of PVM and HRSV.
0001-5573 # 1998 SGM CEBB
P. M. Cook, R. P. Eglin and A. J. Easton
HRSV is the major cause of viral lower respiratory tract proteinase K in 0n1 M Tris-HCl pH 8, 0n05 M EDTA pH 8 (preheated to
disease in infants and young children, causing bronchiolitis and 37 mC) for 8 min at 37 mC and re-ﬁxed in paraformaldeyde. The slides
bronchopneumonia (Aherne et al., 1970). In attempts to were rinsed in 0n1 M triethanolamine (TEA) pH 8, and acetylated in
0n25 % (v\v) acetic anhydride in 0n1 M TEA pH 8 for 10 min at room
understand the mechanisms of pathogenesis of HRSV infection, temperature. After rinsing in two changes of 2i SSC the sections were
several animal models have been used, but none is able to dehydrated by immersion in a series of increasing concentrations of fresh
reproduce the disease seen in human infants (McIntosh & ethanol.
Chanock, 1990). HRSV is able to multiply in the lungs of Virus-speciﬁc probes were prepared from a plasmid containing a full-
several species of rodent under laboratory conditions and, length copy of the PVM N gene inserted into plasmid pGEM-1
despite not being a natural host for HRSV, the mouse has been (Promega) under the control of the SP6 RNA polymerase promoter and
used to evaluate several features of HRSV infection. A model from a 1n08 kbp fragment of the N gene of HRSV strain RS-S2 inserted
into pBluescribe (Stratagene) at the T7 polymerase promoter site. Plasmid
using BALB\c mice is favoured due to its reproducibility, DNA was used as template and negative-sense radioactive RNA probes
among other factors, but the mice show few, if any, signs of were prepared by in vitro transcription in the presence of [$&S]UTP. The
respiratory illness (Taylor et al., 1984). BRSV in cattle has been puriﬁed probes, in buﬀer containing 1 % SDS, 10 mM Tris-HCl pH 7n4,
used as a model for HRSV but this is limited for practical 1 mM EDTA and 10 mM DTT, were added to hybridization buﬀer [50 %
reasons. PVM in mice may oﬀer an amenable model system for (v\v) formamide, 10 % (w\v) dextran sulphate, 1i Denhardt’s solution
the analysis of a mammalian pneumovirus infection in a natural (Sambrook et al., 1989), 150 mM NaCl, 0n5 mM Tris-HCl pH 8, 0n1 M
host, and this model can be compared to the frequently used DTT and 100 µg\ml tRNA]. After heating at 65 mC for 10 min, and
centrifuging at 2000 g for 10 min, 75 µl of the supernatant was placed on
model of HRSV infection in mice. A comparison of the two each slide. A coverslip was placed on the section and the edges were
may indicate diﬀerences which direct the diﬀerent outcomes of sealed. The slides were incubated at 65 mC overnight.
infection. Slides were next washed in 4i SSC, digested with RNase A for
We describe here an analysis of the progress of PVM 30 min at 37 mC to remove excess unbound probe and washed
infection in BALB\c mice by using in situ hybridization to successively twice in 2i SSC, once in 1i SSC and once in 0n5i SSC
follow the location of virus-speciﬁc RNA in the lung, and for 10 min each. All washes contained 1 mM DTT. The slides were
compare this with mice infected with HRSV. The two viruses washed in 0n1i SSC, 1 mM DTT (preheated) at 60 mC for 30 min and
cooled in 0n1i SSC, 1 mM DTT at room temperature. The sections were
exhibited diﬀerences in the distribution of virus RNA in the dehydrated by immersion in an ethanol series containing 0n08i SSC and
lung. 1 mM DTT. After drying the slides, radioactivity was visualized with
liquid emulsion (LM-1 Hypercoat ; Amersham) following the manu-
Methods facturer’s instructions. After 2 weeks the photographic emulsion was
Virus and cells. A pathogenic strain of PVM, J3666, was supplied developed and the sections were stained with haematoxylin and eosin.
by D. Harter (Rockefeller Institute, New York). This strain has been
passaged entirely in mice, and was grown once in BS-C-1 cells to increase Results
the volume of the stock immediately prior to use in the studies reported Characteristics of experimental infections of mice with
here. The A2 strain of HRSV was used to infect mice.
PVM and growth of PVM in the lungs of infected
Infection of animals. Six-week-old speciﬁc-pathogen-free BALB\c
animals
mice were purchased from Harlan Olac (Bicester, UK). The mice were
certiﬁed free of PVM and were transported in ﬁltered cages. Animals In preliminary experiments the clinical signs of infected
were housed in an isolator maintained at a negative pressure of 2n5 mm mice were noted and used to devise a system for assigning a
H O. Mice were lightly anaesthetized with diethyl ether and intranasally score (Table 1). Mice, in groups of 5 to 12, were infected with
#
inoculated with 50 µl virus inoculum on the external nares. After animals
were sacriﬁced one lung from each animal was removed and ﬁxed
immediately by immersion in formal saline. A 10 % (w\v) suspension in Table 1. Scores associated with clinical signs caused by
PBS was generated by Dounce homogenization of the remaining lung, infection of mice with PVM strain J3666
and the suspension was stored at k70 mC. The lower limit of sensitivity
of the assay was 200 p.f.u.\g. For the determination of the clinical
outcome of infection ﬁve mice were infected with 60 p.f.u., 70 with Clinical score Clinical signs
120 p.f.u. and 22 with 600 p.f.u. PVM strain J3666.
In situ hybridization. The ﬁxed lungs were processed overnight in 1 Healthy with no signs of illness
open cassettes on a Shandon Processor model 2LE with standard 2 Consistently ruﬄed fur, especially on neck
processing times. The tissues were embedded in paraﬃn wax on a Tissue 3 Piloerection, breathing may be deeper and mice
Tek III Thermal Console and stored at 4 mC. Sections (4 to 6 µm) were less alert
mounted on glass slides coated with 3-aminopropyltriethoxysilane 4 Laboured breathing. Frequently showing tremors
(Easton & Eglin, 1991). and lethargy
The pre-treatment of sections was based on methods provided by H. 5 Abnormal gait and reduced mobility. Laboured
Goram (Institute of Molecular Medicine, Oxford) and F. Lewis (Institute breathing. Frequently emaciated. May show
of Pathology, University of Leeds). After de-waxing in xylene and cyanosis of tail and ears
rehydration, the sections were post-ﬁxed in 4 % (w\v) paraformaldehyde 6 Death
in PBS for 20 min. The sections were digested in 0n001 % (w\v)
CEBC
Pneumovirus infection in mice
Fig. 1. Effect of quantity of infectious virus on the average clinical scores
of mice following infection with PVM strain J3666. Mice were examined at
daily intervals and clinical scores were assigned, according to the scheme
described in Table 1, following infection with 60 ( ), 120 (
) or 600
(#) p.f.u. in an inoculum volume of 50 µl. Results are expressed as Fig. 3. Replication of PVM strain J3666 in the lungs of infected mice. Titre
means from ﬁve mice. The daily mortality is given as a percentage of the of virus (p.f.u./g) in lung homogenates prepared at daily intervals is shown
number of animals infected with a given dose of virus. following infection of mice with 120 p.f.u. PVM. The line is drawn through
the geometric mean and standard deviations are indicated. The lower limit
of detection of the assay was 200 p.f.u./g.
The volume of inoculum used had a signiﬁcant eﬀect on the
outcome of infection. Fig. 2 shows the results of infection with
three diﬀerent volumes of inoculum containing the same
(120 p.f.u.) dose of virus. As can be seen, reducing the inoculum
volume from 50 to 25 µl diminished the severity and duration
of the clinical signs. Reducing the inoculum volume even
further to 10 µl almost completely abolished signs of infection,
with just one mouse showing mild signs of infection. For
Fig. 2. Effect of inoculum volume on the average clinical scores of mice subsequent experiments mice were inoculated with 120 p.f.u.
following infection with PVM strain J3666. Mice were infected with
120 p.f.u. in an inoculum volume of 10 ( ), 25 (#) or 50 µl (
),
in 50 µl.
examined at daily intervals and clinical scores assigned, according to the The rate of multiplication of PVM in mouse lungs has not
scheme described in Table 1. Results are expressed as means from ﬁve been reported before and it was of interest to determine this
mice.
fundamental characteristic. Following infection, mice were
sacriﬁced on days 2 to 8, day 10 and day 12 and lung
60, 120 or 600 p.f.u. PVM strain J3666 by intranasal homogenates were prepared. The mean titre of each homo-
inoculation, and observed for 15 days. The external observable genate, plotted against time after infection, are given in Fig. 3.
clinical signs of the mice were noted and a mean score for the Virus titres were below detectable levels (less than 200 p.f.u.\g)
animals was calculated on each day (Fig. 1). Depending on the after day 10. The results indicate that the virus replicates
quantity of virus in the inoculum, mice showed the ﬁrst signs eﬃciently in the lung, reaching a peak mean titre of
of illness between days 3 and 5, with laboured breathing, 6n7i10' p.f.u.\g on day 5 post-infection. This peak occurred
wasting and cyanosis at their greatest between days 6 and 9. prior to the highest clinical score achieved with the same dose
Fatalities began to occur on day 6 rising to a maximum on day of virus (Fig. 2).
9, although some deaths occurred as early as day 5 in mice
inoculated with 600 p.f.u. Signs of recovery were seen in the
surviving mice from days 5 to 11, with all mice appearing Detection of PVM RNA in infected mouse lungs by in
healthy after day 12. The onset and severity of disease were situ hybridization
related to the amount of virus in the inoculum, with illness Single lungs from mice infected with 120 p.f.u. PVM strain
occurring earlier in mice infected with the highest dose of J3666 and sacriﬁced on days 2 to 12 post-infection were ﬁxed
600 p.f.u. than in those infected with lower doses. Overall, in formal saline and hybridized with the PVM N gene
similar disease proﬁles were seen for all doses in a 50 µl riboprobe designed to detect virus mRNA and antigenome
inoculum. RNA. In addition, further sections were taken from each
CEBD
P. M. Cook, R. P. Eglin and A. J. Easton
Fig. 4. Detection of pneumovirus RNA by in situ hybridization. Sections from lungs of mice infected with PVM or HRSV were
hybridized with radioactively labelled virus-speciﬁc probes. Signal was detected by autoradiography. Lung sections were
prepared from PVM-infected mice 2 (A ; a hybridizing cell is arrowed), 3 (B), 4 (C), 5 (D) and 8 days (F) post-infection and
hybridized with a PVM-speciﬁc probe. A sample was also prepared from a mouse which had died as a result of PVM infection 6
days post-infection (E). Lung sections were prepared from HRSV-infected mice 5 days post-infection and hybridized with an
HRSV-speciﬁc probe (G–H). Magniﬁcation i400 (A–D, G–H) or i200 (E–F).
CEBE
Pneumovirus infection in mice
sample and hybridized with a probe from the HRSV N gene to
detect non-speciﬁc hybridization. There was no cross-hybrid- Detection of respiratory syncytial virus RNA in infected
ization between the PVM and HRSV N genes under the mice lungs by in situ hybridization
conditions used, and no non-speciﬁc hybridization was seen Six mice were infected intranasally with 1500 p.f.u. HRSV
(not shown). in a 50 µl inoculum volume. Four mice were sacriﬁced on day
Viral RNA was detected in tissue sections as early as day 2 5. At this time the mice had no detectable external clinical signs
post-infection. The dark silver grains, indicating a positive of disease and appeared well. A plaque assay of lung
signal, appeared localized to individual cells, with little homogenates prepared from each mouse gave a mean titre of
background. Most of the positively hybridizing cells were in 4n8i10$ p.f.u.\g, indicating that the virus had replicated
the alveoli (Fig. 4 A), with a considerably smaller number of within the lungs.
cells in terminal bronchioles also showing a positive signal. By In situ hybridization was carried out using the HRSV N
day 3 post-infection the signal was localized in strongly gene probe, with the PVM N gene probe acting as the non-
hybridizing regions while other areas of the lung showed no speciﬁc control probe, and as before no non-speciﬁc hybrid-
hybridization. The number of cells showing a positive signal ization was seen (not shown). The positive signal appeared
had increased when compared with sections from day 2, but predominantly in peribronchiolar and perivascular regions of
the infection was still localized, with individual cells appearing the lungs and was distributed over fairly large areas. Typical
positive while adjacent cells remained negative (Fig. 4 B). examples are shown in Fig. 4 (G–H). While a few terminal
By days 4 and 5 post-infection, when signs of respiratory bronchiolar epithelia and alveolar cells were individually
distress were seen in the animals, the tissue sections showed positive, the overall number was considerably lower than that
morphological changes in the epithelium of some terminal seen in mice infected with PVM on day 5 post-infection.
bronchioles, and in places the epithelium had pulled away from Overall, the pattern of hybridization seen with HRSV-infected
the basement membrane. In these sections eosinophilic material mice was considerably diﬀerent to that seen with PVM-
was frequently seen in the lumen of the bronchioles. Hybrid- infected mice, suggesting that HRSV was restricted in its
ization was seen in cells of the terminal bronchiolar epithelium ability to replicate in certain areas of the lung.
with large areas of some bronchioles, including those showing
morphological abnormalities, giving a very strong signal (Fig.
4 C–D). The hybridization signal in alveolar regions was less Discussion
strictly localized, with a faint signal often seen in cells adjacent Infection of BALB\c mice with as little as 60 p.f.u. PVM
to the alveolar septa. This suggests that the fainter signal may strain J3666 led to a productive infection in which the virus
be the result of secondary infection. In many areas of the grew to high levels with signiﬁcant morbidity and mortality.
sections the deposition of silver grains was concentrated on The external signs of infection ranged from the mildest, where
the apical surface of the terminal bronchiolar epithelium. This piloerection of fur could be seen, to severe pneumonia with
could be seen more clearly when sections were examined by emaciation which was sometimes fatal. The time of onset of
dark-ﬁeld microscopy (not shown). overt disease and of recovery were dependent on the infectious
On day 6 post-infection one mouse died as routine checks dose used, with higher doses leading to earlier onset of clinical
were taking place. Its lungs were excised and ﬁxed im- signs but also to slightly earlier indications of recovery (Fig. 1).
mediately. An unusually strong positive signal was observed This is consistent with earlier analysis of PVM infections in
in up to three-quarters of the area of sections prepared from the mice (Horsfall & Ginsberg, 1951). It is likely that the early
lungs of this animal. All types of alveolar and bronchial cells onset results from a higher virus load reaching the lungs, and
were infected, while the vascular tissue remained uninfected this is consistent with the observation that the inoculum
(Fig. 4 E). The signal was much stronger and more widely volume was also important in determining the outcome of
spread than that seen in mice sacriﬁced on day 5 post-infection, infection, with smaller volumes resulting in milder clinical signs
or in other mice sacriﬁced on day 6 or later. (Fig. 2). The tissues showed clear signs of pneumonia and this
From day 8 post-infection onwards the number of posi- was particularly marked in the terminal bronchioles. This
tively hybridizing cells was considerably less than that seen contrasts with HRSV infection of human infants in which a fatal
earlier, with virus RNA detected only in scattered cells in very outcome has been proposed to be due to blockage of the
few parts of the lung (Fig. 4 F). From day 10 onwards no bronchiolar lumen with squamous plugs, leading to con-
positive signal could be detected by in situ hybridization. At striction of the airways and collapse of the alveolar sacs
this late time-point alveolar tissue in the lungs of some animals (Aherne et al., 1970). It is possible that the choice of mouse
appeared normal, whereas in others there were signs of strain for these studies may inﬂuence the pathological changes
haemorrhage and mononuclear lymphocyte inﬁltration. Simi- seen. For example, it is known that the T-cell response to virus
larly, most of the terminal bronchioles appeared normal while antigens of diﬀerent strains of mice may diﬀer.
a small number of others contained eosinophilic exudate. By Following infection with PVM, in situ hybridization
day 12 post-infection the lungs from all mice appeared normal. detected virus positive-sense RNA, mRNA and antigenome in
CEBF
P. M. Cook, R. P. Eglin and A. J. Easton
cells after 2 days. The number of hybridizing cells in the lungs In tissue samples taken from day 8 onwards the animals had
suggests that by this very early time the virus had already passed the point at which they were likely to succumb to a fatal
undergone one or more rounds of replication, and this is infection, and these animals should be regarded as recovering
supported by the infectivity assay of lung homogenates (Fig. from the infection. At these later times the number of sites at
3). At this time virus was detected most frequently in single which virus RNA was detected was signiﬁcantly reduced when
cells, primarily in the alveoli. Unfortunately, the thickness of compared to those seen on days 5 and 6 (Fig. 4 F). The cells
the sections necessary to ensure a positive hybridization signal showing hybridization to the probe, which were frequently
prevented identiﬁcation of the type of alveolar cell that was single, were scattered throughout the sections, with large areas
infected. As the infection progressed the number of infected of apparently normal uninfected tissue. On day 10 and later no
sites increased, indicating further spread of the infection. The hybridizing cells were detected, indicating that virus clearance
route and mechanism of spread of pneumoviruses from the had occurred to a level below that detectable by in situ
initial site of infection to these presumably secondary sites is hybridization. Some small regions of the tissue sections showed
not known. It is possible that the virus was already present at indications of minor pathological abnormalities but most of the
these sites on day 2, but below the level of detection of in situ tissue was normal, suggesting that tissue damage seen at the
hybridization. The infection was localized in that while some early stages of infection was in the process of being repaired.
areas of the lung showed a number of infected cells there were Mortality occurred at the same time as lung virus titre and
many areas which showed no signs of infection, and this was the number of infected sites began to decline, and this might be
consistent throughout the course of infection in the sections taken to suggest a role for immune-mediated pathology in the
analysed. As the infection continued, the hybridization signal most severely aﬀected animals. Such immune-mediation of
suggested that the virus was able to spread from the initially disease was seen in HRSV-infected mice receiving adoptive
infected cell into adjacent cells, leading to some regions of the transfer of primed CD8+ MHC-I-restricted cytotoxic T cells
lung having very intense signal. The type of cells infected also (Cannon et al., 1988, 1989). Similarly, the potentiation of
altered with time, with some of the columnar epithelia of the HRSV disease by formalin-inactivated virus in cotton rats is
terminal bronchioles becoming infected by days 4 and 5 post- thought to have been immune-mediated (Murphy et al., 1990).
infection (Fig. 4 C–D). The tissues also showed marked PVM-infected athymic mice also die exhibiting clinical signs
histopathological abnormalities, for example nuclei became similar to those described here, and the absence of inﬂam-
acentrically located in some bronchiolar epithelia and in other matory cells in the lungs of PVM-infected mice in this study
regions cells had detached from the basement membrane. The leaves the involvement of the immune system open to question
damage to the bronchiolar epithelia was apparent immediately (Carthew & Sparrow, 1980 b ; Richter et al., 1988 ; Weir et al.,
before the peak in the clinical scores, which assessed the 1988). Once again, diﬀerent strains of mice may give a
severity of the resultant disease (Figs 1 and 2), and these diﬀerent result.
signiﬁcant histopathological changes may be directly as- Infection of mice with HRSV has been used as a model for
sociated with the more severe external signs of disease. In the human infection. It was therefore of interest to compare the
many sections eosinophilic exudate could be seen in the lumen pattern of multiplication of HRSV with that of PVM. Previous
of several terminal bronchioles, and haemorrhaging in the studies have indicated that RSV antigen is found only in the
region of both alveoli and bronchioles was evident in some alveoli, but not the bronchiolar epithelium, of BALB\c mice
mice, consistent with bronchiolitis. The number of sites of (Taylor et al., 1984). A mild cellular inﬁltration of mononuclear
infection continued to rise to a peak on day 6, coinciding with cells and polymorphs was also observed (Anderson et al.,
the peak in lung virus titre. The most extreme example of this 1990). The time-scale of infection of mice with PVM strain
was seen in the lungs of the mouse that died naturally of the J3666 was similar to that reported for HRSV infection ;
infection. In the lung of this animal (Fig. 4 E) there was no however, mice infected with HRSV showed no clinical signs of
signiﬁcant hybridization to cells of the vascular system, despite disease (Anderson et al., 1990 ; Taylor et al., 1984). In our study
very intense hybridization to the adjacent lung tissue. This only a few HRSV-infected alveolar and bronchial epithelium
suggests that haematogenous spread of PVM is unlikely and cells were seen by in situ hybridization. The strongest signal
may explain, in part, the strict pneumotropism of this and was in the peribronchiolar and perivascular regions (Fig.
related viruses. 4 G–H). This was diﬀerent to the situation seen with PVM. In
It is interesting to note that in many bronchiolar epithelial situ hybridization using an HRSV N gene in infected cotton
cells the distribution of silver grains was not random but rats detected virus RNA in both bronchiolar and alveolar
appeared to be preferentially localized at the apical surface. The regions (Murphy et al., 1990). The presence of HRSV in
reason for this is not clear, and while it is tempting to suggest perivascular and peribronchiolar regions has previously been
that this reﬂects budding of virus, as has been reported for seen in samples from human infants (Aherne et al., 1970) and
HRSV in polarized cells (Roberts et al., 1995), the hybridization from calves (Taylor et al., 1984), but these were accompanied
probe detected mRNA and not genomic RNA. The localization by a severe inﬂammatory response, interstitial pneumonia and
was speciﬁc since it was not seen with the control probes. bronchial hyperplasia, which was not seen in BALB\c mice.
CEBG
Pneumovirus infection in mice
This suggests that the use of HRSV in mice as a model for Easton, A. J. & Eglin, R. P. (1991). In situ hybridization. In Methods in
infection of human babies and infants may be of limited use, Gene Technology, vol. 1, pp. 185–202. Edited by J. W. Dale & P. G.
Sanders. London : JAI Press.
possibly because the infection is more restricted.
It is clear that the pattern of HRSV infection in animals is Eaton, M. D. & van Herick, W. (1944). Demonstration in cotton rats and
rabbits of a latent virus related to pneumonia virus of mice. Proceedings of
host-dependent and is diﬀerent to that of PVM in mice. the Society for Experimental Biology and Medicine 57, 89–92.
Although PVM induces severe disease and a productive
Gimenez, H. B., Cash, P. & Melvin, W. T. (1984). Monoclonal antibodies
infection in BALB\c mice, it is uncertain whether PVM to human respiratory syncytial virus and their use in comparison of
infection in mice is analogous to HRSV infection in babies and diﬀerent virus isolates. Journal of General Virology 65, 963–971.
infants. Further work on human infections may help to resolve Homberger, F. R. & Thomann, P. E. (1994). Transmission of murine
this. Investigation of the process of PVM infection in its viruses and mycoplasma in laboratory mouse colonies with respect to
natural host oﬀers an amenable system for further study which housing conditions. Laboratory Animals 28, 113–120.
may identify aspects which are of relevance to HRSV infection Horsfall, F. L. & Curnen, E. C. (1946). Studies on pneumonia virus of
in humans. Of particular interest will be the nature of the mice (PVM). II. Immunological evidence of latent infection with virus in
immune response in infected animals, and the diﬀerences which numerous mammalian species. Journal of Experimental Medicine 83, 43–64.
occur during infection with non-pathogenic PVM strains. Horsfall, F. L. & Ginsberg, H. S. (1951). The dependence of the
pathological lesion upon the multiplication of pneumonia virus of mice.
Journal of Experimental Medicine 93, 139–150.
We would like to thank Dr H. Goram and Dr F. Lewis for help with
the technical aspects of this work, Drs J. Barr, P. Cane and D. Stott for Kraft, V. & Meyer, B. (1990). Seromonitoring in small laboratory animal
providing plasmids used in this work and Professor N. Dimmock for W
colonies. A ﬁve year survey : 1984–1988. Zeitschrift fur Versuchstierkunde
helpful comments on the manuscript. P. M. C. was supported by a 33, 29–35.
research studentship funded by the Biotechnology and Biological Ling, R. & Pringle, C. R. (1989). Polypeptides of pneumonia virus of
Sciences Research Council. mice. I. Immunological cross-reactions and post-translational modiﬁ-
cations. Journal of General Virology 70, 1427–1440.
McIntosh, K. & Chanock, R. M. (1990). Respiratory Syncytial Virus, 2nd
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